DESCRIPTION: Abnormal host-microbial interactions are implicated in the pathogenesis of inflammatory Bowel disease (IBD). Crohn's disease (CD) risk alleles, such as the NOD2 and ATG16L1, have been associated with defective host containment of commensal bacteria. We hypothesize that these CD risk alleles contribute to compositional changes in intestinal microbiota. These changes in microbiota might reduce the proportion of protective commensal organisms and increase the numbers of proinflammatory organisms that incite intestinal inflammation. We propose to take a metagenomic approach towards testing this hypothesis in three Specific Aims: 1.) Analyze the effect of CD-risk alleles on mucosal associated microbiota in GI tissues collected from ileal CD, non-ileal IBD and non-IBD patients;2. ) Develop and test novel genetic probes for pro-inflammatory commensal organisms;3.) Expand prospective collection of GI tissues in four major IBD centers. During the UH2 pilot year, we will focus our study on 755 existing GI tissues from 364 subjects already collected at three institutions. High throughput 16S rRNA sequence analysis of the combined ileal tissues and high throughput genotyping for all highly reproducible CD risk alleles (~30, including NOD2 and ATG16L1) will be performed at Washington University. Quantitative PCR will be performed to quantify specific microbial subgroups. Multivariate analysis of the metagenomic data and the genotyping data will include potentially confounding variables such as age, disease state, medications and smoking. The adherent invasive E. coli (AIEC) are a novel group of bacterial strains that have been implicated as candidate "pathogenic" organisms that incite intestinal inflammation. Because there is limited genome information on AIEC strains, we will produce genome sequences of AIEC strains isolated from the GI tissues. During the UH3 phase of the project, prospective collection of GI tissues from 700-800 ileal CD, non-ileal IBD and non-IBD patients a year will be conducted at four major IBD centers: Washington University, University of North Carolina, Cleveland Clinic, and Mount Sinai Medical Center. Shotgun sequencing will be performed on selected fecal samples linked to the ileal tissues to identify additional, or auxiliary, or synergistic pathogenic factors or other functional changes in the microbiome that are contributed by uncultivatable organisms. Genetic probes for pathogenic commensal organisms developed by mining the bacterial genome data and shotgun sequencing data will be used to test the hypothesis that increased numbers of these organisms are associated with dysbiosis and/or CD risk alleles. Our combined expertise in multiple disciplines across multiple institutions, our demonstrated ability to collect a large number of well-phenotyped samples with longitudinal clinical information that will be linked to host response profiling and morphologic studies, and our consortium's capacity for high-throughput sequencing will be used to investigate how alterations in the human microbiome relate to CD risk alleles and CD pathogenesis. PUBLIC HEALTH RELEVANCE: We have combined expertise in multiple disciplines and the capability to collect a large number of well-phenotyped human clinical samples across six institutions (Washington University in St. Louis, University of North Carolina, University of Colorado, University of New Mexico, Mount Sinai Medical Center, and Cleveland Clinic) with the capacity for high-throughput sequencing at the Genome Center at Washington University to study how alterations in the human intestinal microbiome relate to Crohn's disease.